New antibiotics are needed, particularly those that can be considered as new chemical entities and have novel targets relative to the current, clinical armament of antibiotics. Highly modified nucleoside antibiotics that inhibit bacterial translocase I (TL1) involved in cell wall biosynthesis fit both these descriptions, and have excellent potential in part because they are (i) nanomolar inhibitors of TL1, (ii) inhibit a target that has been proven to be essential for the survival of most, if not all, bacteria, (iii) are effective antibiotics in both in vitro and in vivo models, and (iv) have no apparent toxicity in mice. We have defined the biosynthetic mechanism leading to the core disaccharyl-nucleoside structure of several promising nucleoside antibiotics including A-90289 from Streptomyces sp. SANK 60405, muraminomicin from Streptosporangium sp, and muraymycin from Streptomyces sp. LL-AA896 using a combined in vivo and in vitro approach. The results have defined a multi-enzyme pathway highlighted by divergence from the primary building block UMP and reconvergence to form the core nucleoside. This data was utilized to scan the wealth of genomic information to identify a new lead antibiotic, sphaerimicin, which was isolated and revealed to share the nucleoside core structure but have several unique features including a dihydroxylated piperidine ring of unknown origin. We will now accomplish the following specific aims: (i) to define the mechanism for the attachment of the 3-amino-3-carboxypropyl (3A3CP) moiety that generates the last, shared intermediate in the biosynthesis of A-90289, muraminomicin, muraymycin, and sphaerimicin, which is hypothesized to occur via a new enzyme strategy catalyzed by a pyridoxal-5?-phosphate-dependent protein and (ii) to delineate the biosynthetic mechanism for divergence from the last, shared intermediate to generate unique, nucleoside core scaffolds that are further decorated by fatty acids, polyketides, nonribosomal peptides, and/or saccharides. A biosynthetic mechanism for the diazepanone ring for A-90289 and the highly unusual fused piperidine ring system in sphaerimicin will be defined.